Download Supplementary Information (doc 81K)

1
Supplementary information
2
Physiological investigation
3
Motor nerve conduction studies: for the mother AJG82, median nerve (at elbow): 56.2 m/s,
4
peroneal nerve: at fibular head 52.3 m/s, at the knee 62.1 m/s, tibial nerve (at the knee) 46.1
5
m/s, low CMAP amplitudes and normal conduction velocities; for AFB2, median nerve
6
(proximal): 20.5 m/s, peroneal nerve at the fibular head: 20.0 m/s, tibial nerve (at the knee)
7
14.7 m/s, low CMAP amplitudes and low normal conduction velocities; for AFB3, ulnar
8
nerve R (at wrist) 28.0 m/s, tibial nerve R (at knee) 11.0 m/s, very low CMAP amplitudes
9
(ulnar nerve 27.0%, tibial nerve 5.2%, peroneal nerve 23.5%), exceptionally high voltage
10
required; for AFB4, peroneal nerve proximal motor latency 15.6, conduction 7 m/s, median
11
nerve: distal motor latency 3.8, amplitude 0.283 mV, proximal motor latency 13.6, conduction
12
6 m/s. Needle EMG investigation: for AGJ82, abnormal activity of muscle fibers at rest; for
13
AFB2, no spontaneous activity of muscle fibers at rest; for AFB4, massive spontaneous
14
activity of muscle fibers at rest with no voluntary movements and positive waves suggesting
15
denervation. Muscle biopsy analysis revealed the presence of centralized nuclei for AGJ82
16
and AFB2 but not for AFB4. Altogether, these features are suggestive of centronuclear
17
myopathy for AGJ82, centronuclear myopathy or lower motor neuron disease for AFB2,
18
demyelinating Charcot-Marie-Tooth disease or Spinal muscular atrophy (SMA) for AFB3 and
19
anterior horn disease or SMA for AFB4.
20
21
METHODS
22
Muscle biopsy preparation and staining
23
Muscle biopsies were obtained from AFB2, AFB4 (quadriceps) and their mother AGJ82
24
(tibialis anterior). The AFB4 biopsy was obtained post-mortem. The AFB4 and AFB2
25
biopsies were analyzed with Hematoxin-eosin staining. Ten µm sections were incubated for 1
1
1
h with R2641, rabbit in house polyclonal antibodies against dynamin 2,1 then 45 min with
2
secondary antibodies (AlexaFluor 488 goat anti-rabbit, Invitrogen). Fluorescence was
3
examined with a Leica SP2-AOBS confocal microscope (Leica Microsystems). Pictures were
4
processed with the Tcstk and Dvrtk software (Jean-Luc Vonesch, Imaging Center, IGBMC)
5
and Photoshop 7.0 (Adobe).
6
7
Cell culture
8
Control (3 unrelated cell lines) and patient (AFB2 with the homozygous p.Phe379Val
9
mutation) fibroblasts were grown in Dulbecco’s modified medium (DMEM) supplemented
10
with 10% (v/v) fetal calf serum (FCS) at 37°C in a humidified incubator, 5% CO 2. Protein
11
concentrations were determined as per standard techniques and 30 µg of each sample were
12
resuspended in Laemmli loading dye and analyzed by Tris-Glycine SDS-PAGE and western
13
blot analysis. Antibodies employed included GAPDH (Millipore), and in house R2680 rabbit
14
polyclonal anti-dynamin 2 antibodies.1
15
16
Transferrin uptake
17
Serum-deprived fibroblasts were incubated with AlexaFluor 633 conjugated transferrin
18
(Invitrogen) for 15 min. Cells were washed twice for 3 min in 0.2 M acetic acid, 0.5 M NaCl
19
followed by washing in 0.25 M Tris (pH 10) for 2 min and were subsequently fixed in 1%
20
(v/v) formaldehyde. To block dynamin action, cells were treated with dynasore, a small non-
21
competitive inhibitor of dynamin by acting on its GTPase domain (Sigma Aldrich).2 The
22
samples were analyzed on the FACSCalibur (BD Biosciences) employing the Cell Quest Pro
23
program (BD Biosciences). Subsequent analysis was performed employing the Flowjo
24
software (Tree Star Inc., Oregon, USA). The student’s t test was used for statistical analysis. P
25
values of <0.01 were considered significant.
2
1
Dynamin 2 in vitro experiments
2
Cloning, expression and purification: The cloning of the wild-type human dynamin 2 (isoform
3
1; Accession number NM_001005360) into pENTR1A has been reported elsewhere.3 Human
4
dynamin 2 mutant (p.Phe379Val) was generated by primer directed PCR mutagenesis from
5
the wild type construct using primers 5’ CTGGTAAAGATGGAGGTTGACGAGAAGGAC
6
3’ and 5’ GTCCTTCTCGTCAACCTCCATCTTTACCAG 3’. The pDEST8 (Invitrogen)
7
expression constructs were generated by homologous recombination. WT and mutant
8
(p.Phe379Val) human DNM2 were expressed in SF9 insect cells and purified against GST-
9
SH3 (Amphiphysin I; a kind gift from P. De Camilli). Purified dynamin 2 proteins were
10
stored in 20 mM HEPES-KOH pH 7.4, 300 mM NaCl, 1 mM DTT, 1 mM EGTA, 30% (v/v)
11
glycerol, snap frozen and stored at -80°C. GTPAse assays to measure dynamin’s basal
12
activity were performed in 20 mM HEPES-KOH 7.4, 100 mM NaCl, 1 mM MgCl2 using the
13
Malachite green method in the presence of 0.5 mM GTP. 4 EM tubulation assays: 2-5 µL of
14
GTPase buffer (20 mM HEPES pH 7.4, 100 mM NaCl, 1mM MgCl2) were deposited on
15
parafilm to form a water droplet. Purified dynamin 2 protein solution (2-5 µl of a 0.5-2 mg/ml
16
solution) were then added to the droplet, and the reaction was initiated by adding 0.5-1µL of
17
liposomes composed of 85 % (m/m) phospholipids [30% brain phosphatidylethanolamine
18
(PE), 5% liver phosphoinositides (PI), 30% palmitoyl-oleyl phosphatidylserine (POPS) and
19
35% palmitoyl-oleyl phosphatidylcholine (POPC)], and 15% (m/m) cholesterol; Avanti Polar
20
Lipids Inc., USA prepared using the spontaneous growth method.5 After 5 min incubation at
21
room temperature, a carbonized EM grid was deposited on the top of the droplet for 1 min.
22
The grid was then washed in a 50 µL droplet of GTPase buffer, blotted against filter paper,
23
and stained by depositing the grid onto a 10 µL Uranyl Acetate 2% droplet for 30 sec. Grids
24
were then dried with filter paper, and directly analyzed with a FEI Tecnai G2 Sphera
25
microscope.
3
1
2
In situ hybridization
3
In situ hybridization assays on mouse embryos sections were performed, employing the Dnm2
4
mRNA probe (obtained from Eurexpress, http://www.eurexpress.org/), as previously
5
described.6 Controls (lacking mRNA probe) were performed in parallel to monitor levels of
6
non-specific binding.
7
8
Injection of morpholinos into zebrafish embryos
9
A translation blocking morpholino (5’ ATTCCTCCATCCCCCGGTTGCCCAT 3’) against
10
the Danio rerio dnm2 mRNA was designed and purchased from Gene Tools (Oregon, USA).
11
Wild-type (AB) and transgenic Tg(flk1:eGFP) zebrafish were used. dnm2 MO (4 or 5 ng) was
12
injected into one cell stage embryos. Anti-acetylated tubulin labeling in zebrafish was
13
performed as previously described. 7
14
15
Imaging and data processing
16
Live zebrafish embryos were anesthetized in Tricaine solution and mounted in low melting
17
point agarose gel. The excitation light was provided by a mode-locked Ti: Sapphire laser
18
(Chameleon Ultra, Coherent Inc., USA) that delivers 150 fs pulses with 80 MHz repetition
19
rate. The excitation beam was focused on the sample by 20x 0.95 N.A. water immersion
20
objective (Leica Microsystems). The excitation wavelength was 900 nm for second harmonic
21
generation. The second harmonic and fluorescence signals were collected in trans and epi
22
configuration respectively. The data visualization was performed using ImageJ and Imaris
23
software (Bitplane).
24
4
1
2
3
4
5
REFERENCES
1.
Cowling BS, Toussaint A, Amoasii L et al: Increased expression of wild-type or a centronuclear
myopathy mutant of dynamin 2 in skeletal muscle of adult mice leads to structural defects
and muscle weakness. Am J Pathol 2011; 178: 2224-2235.
2.
Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T: Dynasore, a cellpermeable inhibitor of dynamin. Dev Cell 2006; 10: 839-850.
3.
Nicot AS, Toussaint A, Tosch V et al: Mutations in amphiphysin 2 (BIN1) disrupt interaction
with dynamin 2 and cause autosomal recessive centronuclear myopathy. Nat Genet 2007;
39: 1134-1139.
13
14
15
4.
Leonard M, Song BD, Ramachandran R, Schmid SL: Robust colorimetric assays for dynamin's
basal and stimulated GTPase activities. Methods Enzymol 2005; 404: 490-503.
16
17
18
5.
Takei K, Haucke V, Slepnev V et al: Generation of coated intermediates of clathrin-mediated
endocytosis on protein-free liposomes. Cell 1998; 94: 131-141.
6.
Schaeren-Wiemers N, Gerfin-Moser A: A single protocol to detect transcripts of various types
and expression levels in neural tissue and cultured cells: in situ hybridization using
digoxigenin-labelled cRNA probes. Histochemistry 1993; 100: 431-440.
7.
Colantonio JR, Vermot J, Wu D et al: The dynein regulatory complex is required for ciliary
motility and otolith biogenesis in the inner ear. Nature 2009; 457: 205-209.
6
7
8
9
10
11
12
19
20
21
22
23
24
25
26
27
28
29
30
31
32
FIGURE LEGENDS
33
the right arm of AFB3 shows slender bones of humerus, radius and ulna with normal bone
34
density.
Supplementary Figure 1. Clinical characterization of patients. Anteroposterior radiograph of
35
36
Supplementary Figure 2. Homozygosity by descent for the five children employing a 250 K
37
Affymetrix Mapping array. Homozygous regions are depicted in dark blue over the whole
5
1
chromosome 19. The largest common region of homozygosity for the three affected children
2
(indicated by a red box) was found on chromosome band 19p13, from 8483868 to 12350726
3
(hg18 release) and contained 141 genes including the DNM2 gene.
4
5
Supplementary Figure 3. Analysis of a muscle biopsy from a heterozygous carrier. (a)
6
Hematoxin-eosin staining of right tibialis anterior muscle biopsy of the AGJ82 heterozygous
7
mother (left: 10x magnification, right: 40x magnification). Heterogeneous fibers size with
8
hypertrophic and hypotrophic fibers and a high proportion of central nuclei are observed,
9
typical for DNM2-related autosomal dominant centronuclear myopathy. Scale bar represents
10
50 µm. (b) The presence of the p.Phe379Val mutation at the heterozygous state does not
11
affect dynamin 2 localization in muscle. Immunostaining of longitudinal sections of control
12
(46 years old) and AGJ82 muscle biopsies with anti-dynamin 2 R2641 specific antibodies.
13
Scale bar represents 10 µm.
14
15
Supplementary Figure 4. Impact of the p.Phe379Val on cellular processes involving DNM2
16
action. (a) The p.Phe379Val mutation does not affect centrosome cohesion as revealed by
17
staining control and p.Phe379Val fibroblasts with γ tubulin antibodies; centrosome cohesion
18
was perturbed in 2%+/-1% of control cells versus 3%+/-1% for p.Phe379Val cells. (b) The
19
p.Phe379Val mutation does not cause trans or cis-Golgi network fragmentation. Trans and
20
cis-Golgi network morphology was compared in control and patient fibroblasts by
21
immunofluorescence using anti-golgin 97 and GM130 antibodies. Nocodazole treatment
22
resulted in fragmentation of the network in both instances. Nuclei were stained with Hoechst.
23
Scale bars represent 10 µm.
24
6
1
Supplementary Figure 5. Dnm2 morphant morphology. Dnm2 morphant phenotype at 48
2
hpf (hours post-fertilization) (a-c) and at 72 hpf (d, e). Dnm2 morphants display variable
3
phenotypes and severe tail truncation can be seen in a small fraction (10%, n=300) of injected
4
embryos (c). These embryos were discarded and further analysis was performed at 72 hpf
5
using non-truncated embryos (70%, n=300) (d, e).
6
Supplementary Figure 6. Morphology of nerves in the tail of control and dnm2 MO-injected.
7
Peripheral nerves detected with anti-acetylated tubulin immunofluorescence (Ac. Tub.) appear
8
similar in control and dnm2 MO embryos.
9
7